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Laboratory of Molecular Biophysics
Laboratory Journal 2002
Declan A. Doyle
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Contents.
Declan A. Doyle
Ion channels and transporters
Declan A. Doyle, Jonathan Cuthbertson, Anling Kuo,
Tahmina Rahman, Jochen Zimmer
Two major hurdles must be overcome if the structure of
a membrane protein is to be determined. The first involves obtaining protein
in sufficient quantities to be able to screen for suitable crystals.
Membrane proteins that are naturally abundant are good targets such as those
involved in the respiratory chain or photosynthesis. Alternatively,
the target protein can be over-expressed in expression systems ranging from
bacteria to mammalian cells. To date, only the Escherichia coli expression
system has been successful in providing membrane protein in sufficient quantities
and in a homogeneous state that has lead to structural determination (Chang
et al., 1998; Doyle et al., 1998; Dutzler et al., 2002).
The next problem is finding a crystal that is sufficiently well ordered and
diffracts to a limit that would make the project feasible. For membrane proteins
this can be a real problem because of the flexible, dynamic detergent micelle
that surrounds the protein. The detergent micelle is necessary for the maintenance
of structural integrity of the membrane protein outside of a phospholipid
bilayer. Generally, for protein crystallography, large degrees of flexibility
within the protein are considered detrimental for high resolution crystals.
To overcome this problem with soluble proteins alternative constructs that
explore domains within the structure or homologous proteins can be considered.
Repeating this method with membrane proteins does not improve the situation
as in all cases the detergent will still be present.
A number of methods have been developed to overcome the
detergent problem in membrane protein structure determination. Increasing
the soluble protein surface area with the use of antibody fragments is one
approach. This method is believed to improve the quality of the crystal by
increasing the number of protein-protein contacts within the lattice and
decreasing the weaker and less stable protein-detergent or detergent-detergent
contacts. In the bicontinous cubic phase method the detergent is no longer
required during the crystallisation step as the protein is re-introduced into
a membrane bilayer environment (Landau and Rosenbusch, 1996).
During our structural studies of the E. coli membrane
protein yadQ we uncovered a novel method for improving the diffraction limits
and integrity of a membrane protein crystal (Kuo, et al, 2002). The improvement
was brought about by the simple process of dehydration. Slow evaporation
of water over a period of months resulted in the improvement in the resolution
limits from 8.0 to 4.0 Å with a concurrent decrease in the mosaicity
to values of approximately 1° (Figure 1).
YadQ is a member of the voltage-dependent chloride channel family. The channel,
as for all others within this family, forms homodimers as the active complex
within a membrane. It has been shown functionally for the eukaryotic
homologue of the bacterial chloride channel that each monomer has a separate
ion conduction pathway that opens and closes independently (Middleton et
al., 1996). This mechanism is referred to as the fast gate. Another gating
mechanism shuts both monomers simultaneously, referred to as the slow gate.
It is conceivable that a conformation change occurs at the dimer interface
during slow gating. We can only speculate as to the exact conformation change
that the channel undergoes upon gating but such a change may account for
the variation in unit cell dimensions that we observe upon dehydration. Equally
as likely is the possibility that the molecule is relatively rigid and the
dehydration process causes the channel to go through a rigid-body rotation.
This may be aided by the presence of the flexible detergent micelle. In this
scenario, the micelle changes its structure during the dehydration process
allowing chloride channel to rotate.
Heavy metal ATPases use the hydrolysis of ATP to move specific ions against
their concentration gradient. It is believed that all of the members within
the ATPase family follow a similar reaction scheme to that of the calcium-ATPase.
This involves binding of ions and ATP followed by phosphorylation of the
enzyme. The ATPase then undergoes a conformational change resulting in the
release of the ions. Dephosphorylation of the enzyme completes the cycle
and the protein sets up to once again accept ions and ATP. Many detailed studies
have characterised the various steps in this cycle and have demonstrated that
for optimum activity the enzyme requires particular closely associated lipids.
We have begun to characterise the lipid requirements of the zinc-ATPase. This
protein has a central core that is structurally related to the calcium ATPase
based on sequence alignments. Initial studies have indicated that,
like the calcium ATPase, the zinc enzyme's activity is enhanced in the presence
of specific lipids. Further studies will be aimed at uncovering the specific
lipid nature of the enzyme activity and domain movement as the protein goes
through the catalytic cycle.
It is believed that the distinct environment that a membrane protein resides
in dictates the type of structure that is formed within the bilayer region,
i.e. only transmembrane alpha helices or only beta sheet. Several sophisticated
transmembrane helical prediction programs are publicly available that identify
potential regions within the linear amino acid sequence that span the membrane.
At present we are analysing these predictions against the known membrane
structure database. This information should provide a clearer picture of
the ability to correctly predict transmembrane helical segments. It
is hoped that with this knowledge experimentalists can fragment the target
protein into it's structural components with a greater degree of confidence.
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Figure 1. Diffraction patterns and
morphologies of crystals of the integral membrane protein yadQ ...more
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References
Chang, G., Spencer, R. H., Lee, A. T., Barclay, M. T., Rees, D. C. (1998)
Structure of the MscL homolog from Mycobacterium tuberculosis: a gated
mechanosensitive ion channel. Science. 282, 2220-2226.
Doyle, D. A., Morais-Cabral, J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M.,
Cohen, S. L., Chait, B. T., MacKinnon, R. (1998) The structure of the potassium
channel: molecular basis of K+ conduction and selectivity. Science.
280, 69-77.
Dutzler, R., Campbell, E. B., Cadene, M., Chait, B. T., MacKinnon, R. (2002)
X-ray structure of a ClC chloride channel at 3.0 Å reveals the molecular
basis of anion selectivity. Nature. 415, 287-294.
Kuo, A., Bowler, M. W., Zimmer, J., Antcliff, J. F., Doyle, D. A. (2002)
Increasing the diffraction limits and internal order of a membrane protein
crystal by dehydration. J. Struct. Biol. In press.
Landau, E. M., Rosenbusch, J. P. (1996) Lipidic cubic phases: a novel concept
for the crystallization of membrane proteins. Proc. Natl. Acad. Sci.
93, 14532-14535.
Middleton, R. E., Pheasant, D. J., Miller, C. (1996) Homodimeric architecture
of a ClC-type chloride ion channel. Nature. 383, 337-340.
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